Method of determining bacterial number of dust in buildings

FIELD: chemistry.

SUBSTANCE: method involves collecting dust samples with a sterile cotton wool wad from 10-20 cm2 of any surface or the sterile cotton wool filter of a vacuum cleaner. The dust samples are put into a sterile physiological solution, followed by extraction for 30-60 minutes at room temperature while stirring. The supernatant liquid is collected and multiple cultures of the obtained extract are prepared from the liquid. The obtained cultures are sown in a semi-liquid thioglycol medium and modified thioglycol medium obtained by adding defibrinated blood to the thioglycol medium in a given amount. Culturing is performed for 10 days at temperature 37C and daily visual monitoring, followed by determining the number of microbial cells which are calculated from the McCready probability table, which is compiled based on data on growth properties of microorganisms and tinctorial properties of the bacterial community of the dust sample are determined through microscopic examination of smears of the mixed culture mass which is gram-stained.

EFFECT: simultaneous quantitative determination of the total bacteria number and obtaining cultural, morphological, tinctorial and haemolytic characteristics of the bacterial community of dust samples from buildings.

 

The invention relates to the field of ecology, namely, to obtain the initial characteristics of the microbial community dust areas, and can be used for monitoring residential and industrial premises with the aim of simultaneous quantitative determination of total bacterial contamination and receiving cultural, morphological, tinctorial and hemolytic characteristics of the microbial community samples of dust areas.

A known method for determining bacterial contamination Cup method through the consistent use of a set of special environments and cultivation under anaerobic and aerobic conditions (instructions for bacteriological control of complex sanitary-hygienic measures in health care institutions. Order No. 720 USSR Ministry of health, dated July 31, 1978, Appendix 2. HOWTO MUK 4.2.734-99. Microbiological monitoring of the working environment. The MOH of Russia. Moscow, 1999 I. Goh, Obbard J.P., Viswanathan, S., Huang Y. Airborne bacteria and fungal spores in the indoor environment. A case study in Singapore. Acta biotechnol., 2000, 20, 1, 67-73).

However, for screening studies of microbial contamination dust areas consistent use of several special nutrient media in different culture conditions greatly increases the effort and time, there is a need for the mine to obtain qualitative characteristics of bacterial flora.

The problem to which this invention is directed, is to develop a method which involves the use of monitoring environmental monitoring determination of bacterial contamination dust areas sowing dust sample on only one nutrient medium and simultaneously obtaining qualitative and quantitative characteristics of the microbial community.

The technical result of the claimed method is the achievement of efficiency, simplicity and convenience in determining the total bacterial contamination of the dust of the premises.

To achieve the technical result of the method of determination of bacterial contamination of the dust of the premises involves collecting samples of dust sterile cotton swab with 10-20 cm2any surface or on a sterile cotton filter when vacuuming, hanging dust or cotton swab in 1 ml of sterile physiological solution, extraction for 30-60 minutes at room temperature with stirring, the selection of the supernatant liquid, the preparation of supernatant multiple breeding extract, seeding the resulting dilution in thioglycolate environment and modified thioglycolate medium prepared by adding to 10 ml of thioglycolic environment defibrinating blood in quantities of 0.2-0.4 ml, stump the licensing within 10 days at a temperature of 37C, daily visual inspection and then determining the most probable number number of microbial cells.

The determination of the most probable number number of microbial cells is calculated by the statistical table Mac-credit, based on the data on the growth properties of microorganisms, and tinctorial properties of bacterial community sample of dust detected by smear microscopy mixed culture of mass, gram-stained.

Preparation of thioglycolic environment. A portion of the environment to control the sterility of thioglycolic environment (thioglycolate Wednesday FS 42-326-SU-92: hydrolyzed casein enzymatic - 15 g, fodder yeast extract 5 g, sodium chloride and 2.5 g, glucose 5 g, thioglycolate sodium - 0.5 g cysteine hydrochloride, 0.75 g, agar-agar microbiological - 0.75 g, sodium carbonate and 0.8 g distilled water to 1 l, pH 7.0) was dissolved by heating in distilled water, poured into 10 ml sterile test tubes and sterilized. To prepare the modified environment to detect the presence of hemolytic microorganisms to 10 ml sterile thioglycolate environment at a temperature of 37-40C, add 0.2-0.4 ml defibrinating blood (animal or human), stirred in a circular motion to obtain a uniformly colored solution.

The samples were collected dust can be the t to be made sterile cotton swab with 10-20 cm 2any surface or collected on sterile cotton filter when processing the surface of the vacuum cleaner.

Preparation of dilution of the extracts of samples of dust. A portion of dust or cotton swab placed in a sterile tube, add a known volume of sterile saline solution, the extraction is carried out for 30-60 minutes at room temperature with stirring, select the supernatant, from which it is prepared dilution of the extract.

Seeding each dilution is carried out in three parallel tubes prepared by the environment, and for planting the minimum breeding use tubes with a modified environment.

Visual inspection allows to make a conclusion about the presence in the microbial community of bacteria with different type of breathing, character growth, ability to flatulence, hemolytic properties.

The most probable number is the number of microbial cells (NWCC) is determined by the table Mac-credit.

Preparation of smears. Smears prepared from thoroughly mixed culture of mass of the tubes with a minimum dilutions, stained by Gram. When using the microscope determine tinctorial properties of the mixed culture is the presence of gram-positive and gram-negative coccoid and rod-shaped bacteria.

The invention is illustrated in SL is blowing examples.

Example 1. Determination of bacterial contamination of dust accommodations

A sterile cotton swab was collected dust with 10 cm2the surface of the plinth. The swab was placed in a sterile tube, add 1 ml of sterile physiological solution. After 30 minutes of stirring has prepared a five-fold dilution of the extract, spent sowing 100 l of each dilution in three parallel tubes topicalias medium and 100 l of minimum cultivation in three parallel tubes with the modified thioglycolate medium prepared by adding to 10 ml of thioglycolic environment 0.2 ml defibrinating blood. Were cultured for 10 days under aerobic conditions at a temperature equal to 37C. Visual inspection revealed the presence of microbial community sample of dust, mainly aerobic microorganisms, as in the upper part of the column environment noted a strong turbidity on the surface of the film growth, at a height of 1 cm from the surface of the medium was observed 2-11 loose bars, the lower part of the environment was clear. Gasification has not been identified as of hemolysis. Microscopic analysis of the mixed culture showed the presence of coccoid and rod-shaped polymorphic bacteria gram-negative and gram-positive. The overall rate was 150 NVCC/cm2.

Example 2. Determination of bacterial contamination of dust collected mattress

A sample of dust collected on sterile cotton filter when processing the surface of the vacuum cleaner. A portion of the dust with a mass equal to 5 mg, was placed in a sterile tube, add 1 ml of sterile physiological solution. After 60 minutes of mixing has prepared a five-fold dilution of the extract, spent sowing 100 l of each dilution in three parallel tubes with thioglycolate medium and 100 l of minimum cultivation in three parallel tubes with the modified thioglycolate medium prepared by adding to 10 ml of thioglycolic environment 0.4 ml defibrinating blood. Were cultured for 10 days under aerobic conditions at a temperature equal to 37C. Visual inspection revealed the presence in the microbial community of the sample dust bacteria with different types of respiration - aerobic and anaerobic (surface and bottom character growth), since the surface environment was noted film growth at the bottom of the tube was observed hemolysis. Microscopic analysis of the mixed culture showed the presence of coccoid and rod-shaped polymorphic bacteria gram-negative and gram-positive. The overall rate was 2105NVCC/g of dust.

Thus, the claimed method simultaneously determined the number of total bacterial contamination, cultural, morphological, tinctorial and hemolytic characteristics of the microbial community samples of dust areas is a simple, effective and convenient for routine studies.

1. The method for determining bacterial contamination dust areas, including dust samples were collected with a sterile cotton swab with 10-20 cm2any surface or on a sterile cotton filter when vacuuming, hanging dust or cotton swab in 1 ml of sterile physiological solution, extraction for 30-60 min at room temperature with stirring, the selection of the supernatant liquid, the preparation of supernatant multiple dilution of the extract, the planting of the resulting dilutions in thioglycolate environment and modified thioglycolate medium prepared by adding to 10 ml of thioglycolic environment defibrinating blood in quantities of 0.2-0.4 ml, grown for 10 days at 37C, daily visual inspection and then determining the most probable number number of microbial cells.

2. The method according to claim 1, characterized in that the determination of the most probable number number of microbial cells is calculated by the statistical table Mac-credit, based on the data on the growth properties of microorganisms, and tinctorial the basic properties of the bacterial community of the dust sample is detected by smear microscopy mixed culture of the masses, gram-stained.



 

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2 tbl

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